Kupyaphores─Self-Assembling Diisocyanolipopeptide Zn ^II Ionophores in <i>Mycobacterium tuberculosis</i> Zn ^II /Cu ^I/II Homeostasis and Antibacterial Effects

<i>Mycobacterium tuberculosis</i> (<i>Mtb</i>), the leading cause of infectious disease mortality from a single pathogen, requires essential metal ions to establish infection and persist in the host. Kupyaphores, a suite of recently identified amphiphilic diisocyanolipopeptides, were reported to assist with Zn^II acquisition to support a multitude of Zn^II-dependent metalloenzymes critical for <i>Mtb</i>'s survival and pathogenicity. However, compared to well-studied Fe^III acquisition systems in <i>Mtb</i>, the mechanisms for Zn^II acquisition and homeostasis remain virtually unexplored. Herein, we reveal them as novel metal ionophores in <i>Mtb</i>'s metal-fluctuating lipidic niche. A concise modular scalable synthesis was developed to assess the critical features required for activity. Synthetic kupyaphores were structurally and functionally validated, respectively, via LCMS and chemical complementation of kupyaphore-deficient (Δ<i>rv0101</i>) <i>Mtb</i>. MS, NMR, and IR evidence demonstrated that kupyaphores complex Zn^II as a bidentate ligand. Fluorescence competition data indicated Zn^II/Cu^I/II binding capabilities, by which <i>Mtb</i> entraps excessive metals within o/w-type micelles against host-induced metal intoxication. The inhibition against Gram-positive <i>Staphylococcus aureus</i> and the low human toxicity imply the potential as a novel antibacterial scaffold. Collectively, this work provides insight into the Zn^II/Cu^I/II homeostasis of <i>Mtb</i> and a chemical basis for the development of mechanistic tools, therapeutic conjugates against <i>Mtb</i>, and antibiotics.